Type 2 diabetes (T2D) has reached epidemic proportions, with ~9.4% of the US adult population being diabetic, and another 84.1 million have pre-diabetes. It is currently acknowledged that both insulin resistance and b-cell dysfunction are early and essential events in the development of T2D. The formation of the SNARE (Soluble NSF Attachment Protein Receptor) complex is rate limiting for insulin secretion. Our understanding of factors that regulate the formation of the SNARE complex and how they contribute to reduced insulin secretion from b- cells in impaired glucose tolerance is lacking. To this end, by using forward genetics approach, we have identified Tomosyn-2, which is an endogenous inhibitor of insulin secretion and functions by binding to syntaxin. Syntaxin is a key component of the SNARE complex that modulates the fusion of the insulin granules to the plasma membrane for insulin secretion from b-cells. We have discovered that a gain-of-function mutation in the Tomosyn-2 gene led to an increase in islet Tomosyn-2 protein abundance and formation of hypoinsulinemic/ hyperglycemic phenotypes in mice. Increased abundance and/or the functional activity of Tomosyn-2 causes reduction in insulin secretion from human and mouse islets. Thus, the long-term goal is to understand how Tomosyn-2 function in b-cells can be manipulated to improve insulin secretion in impaired glucose tolerance for the treatment and prevention of prediabetes and T2D. The objective of this application is to determine how Tomosyn-2 inhibits insulin secretion from b-cells in the pathophysiology, physiology, and at the molecular level, and how its inhibitory function in b-cells is regulated. Our data show that the improved glucose tolerance in Tomosyn-2-null mice is a direct result of enhanced insulin secretion from pancreatic islets. Further, reduced insulin secretion is observed in islets of mice on a high-fat diet that have elevated Tomosyn-2 protein levels. We have identified phosphorylation sites in response to major b-cell signaling pathways that modulate Tomosyn-2 inhibitory function. Also, E3-ubiquitin ligase, Hrd1 and an insulin granule protein, Syt9 bind and regulate the protein abundance of Tomosyn-2. Our hypothesis is that Tomosyn-2 is a key protein in the exocytotic machinery that regulates SNARE complex-mediated insulin secretion in response to nutritional and genetic cues, and that specific post-translational modifications of Tomosyn-2 increase insulin secretion. To test this hypothesis, we propose three aims: 1) determine the sub-cellular mechanisms by which Tomosyn-2 inhibits insulin secretion, 2) determine Tomosyn-2 phosphorylation regulates its activity on downstream insulin secretion, and 3) determine the role of the Tomosyn-2-binding proteins, Syt9 and Hrd1, in regulating insulin secretion. Outcomes from this project will provide novel information on how b-cells prevent inappropriate insulin secretion, identify the molecular target for the early phase insulin secretion, and insights into the loss in fusion competency of insulin granules during impaired glucose tolerance. Our results will provide fundamental new knowledge of the nutritional and hormonal regulation of the SNARE complex, identifying steps that could be modulated therapeutically in T2D.